It is known in the art that homocysteine thiolactone, a metabolite of the essential amino acid, methionine, is metabolized abnormally in malignant cells, as reported in Cancer Research 36:3198-3202, 1976. Because malignant cells are unable to convert homocysteine thiolactone to sulfate ester, excess homocysteine thiolactone accumulates within the cells and reacts with the free amino groups of proteins, nucleic acids, glycosaminoglycans and other molecules, a reaction known as thiolation. Extracts of human malignant tumors contain free homocysteine thiolactone, but normal human tissues contain only trace amounts of the compound, as reported in Research Communications in Chemical Pathology and Pharmacology 59:107-119, 1988. The accumulation of homocysteine thiolactone within cultured malignant cells was shown to be catalyzed by an error-editing reaction that is dependent upon methionyl t-RNA synthase, as reported in FEBS Letters 317:237-240, 1993.
Malignant cells are also known to possess other characteristic abnormalities of methionine metabolism, as reviewed in Annals of Clinical and Laboratory Science 24:27-59, 1994. These abnormalities are (1) poor growth of cultured malignant cells in methionine-free media containing homocysteine and a methyl donor, (2) decreased synthesis of adenosyl methionine and increased synthesis of adenosyl homocysteine in malignant cells, (3) increased transmethylase activity in tumor tissue, (4) hypomethylation of DNA from tumor tissue, (5) prevention by methionine of carcinogenesis in mammals by ethionine or by a choline-deficient diet, and (6) inhibition of aerobic glycolysis of cultured malignant cells by growth in media with a high methionine concentration.
The cause of abnormal homocysteine thiolactone metabolism in malignant cells was suggested to be a deficiency of or failure to form an N-substituted derivative of homocysteine thiolactone, as discussed in Cancer Research 36:3198-3202, 1976. According to this concept, the function of this N-substituted homocysteine thiolactone derivative in normal cells is to prevent accumulation of homocysteine thiolactone, thereby preventing thiolation of free amino groups of proteins, nucleic acids, and glycosaminoglycans, and also thereby preventing the malignant growth state that is characteristic of cancer cells.
The identity of the N-substituted antineoplastic derivative of homocysteine thiolactone that prevents homocysteine thiolactone formation in malignant cells was elucidated by synthesis of the antineoplastic derivatives of homocysteine thiolactone, N-maleyl homocysteine thiolactone amide, N-maleamide homocysteine thiolactone amide, and rhodium trichloride oxalyl homocysteine thiolactone amide, as taught in U.S. Pat. No. 4,383,994. These results show that the antineoplastic homocysteine thiolactone derivative is (1) active in a lipid-soluble form, (2) contains a conjugated double bond system with a carbonyl group adjacent to the nitrogen atom of homocysteine thiolactone, and (3) forms a complex with a transition metal atom that enhances antineoplastic activity. As taught in U.S. Pat. Nos. 4,618,685 and 4,925,931, incorporated herein by reference thereto, homocysteine thiolactone reacts with retinoic acid to form N-homocysteine thiolactonyl retinamide (NHTR), known as thioretinamide, and thioretinamide reacts with cobalamin to form N-homocysteine thiolactonyl retinamido cobalamin (NHTR).sub.2 Cbl, known as thioretinaco. Both thioretinamide and thioretinaco have anticarcinogenic and antineoplastic activities, as reported in Carcinogenesis 8:1559-1562, 1987 and Proceedings of the Society for Experimental Biology and Medicine 191:346-351, 1989.
In experiments with cultured malignant and normal cells, thioretinaco was found to have antiproliferative activity, and thioco, the complex of homocysteine thiolactone and cobalamin, was found to increase growth of both malignant and normal cells, as reported in Research Communications in Chemical Pathology and Pharmacology 77:125-128, 1992. Treatment of rabbits with thioretinamide or thioretinaco, while consuming an atherogenic diet, significantly increased the serum concentration of homocysteine and enhanced atherogenesis, as reported in Atherosclerosis 83:197-206, 1990. Intra-tumor administration of thioretinaco decreased the growth of human pancreatic adenocarcinomas in athymic mice, as reported in Research Communications in Chemical Pathology and Pharmacology 66:117-122, 1989.
Interferons alpha, beta and gamma are cytokines that are know to have antineoplastic, antiviral, and antiproliferative activities, as discussed in Interferons. A Primer. by R. M. Friedman, Academic Press, New York, 1981. Recombinant interferon gamma increases the intracellular content of adenosyl methionine in cultured macrophages, as reported in Journal of Immunology 136:2596-2604, 1986. In that study, the enhanced tumoricidal activity of macrophages activated by interferon gamma was attributed to increased intracellular adenosyl methionine. Methionine and retinoic acid enhance the antiproliferative effects of interferons alpha and beta in transformed and malignant cultured cells, as reported in Journal of Biological Regulators and Homeostatic Agents 2:45-49, 1988.
Treatment of blood with ozone increases the production of interferon gamma by leukocytes, as reported in Haematologica 75:510-515, 1990. Induction of interferon by poly I:C in mice exposed to ozone reduces the extent of lung damage, and anti-interferon antibody increases lung damage, showing an antioxidant activity of interferon, as reported in Toxicology Letters 39:51-62, 1987. Interferon therapy of human patients with hepatitis C infection decreases the concentration of serum lipid peroxidation products by an antioxidant effect, as reported in Free Radical Biology and Medicine 16:131-133, 1994.
Ozone selectively inhibits the growth of human cancer cells in culture, compared to its effect on normal human lung diploid fibroblasts, as reported in Science 209:931-933, 1980. Ozone decreases the carcinogenicity of urethan in producing pulmonary tumors in mice in a dose dependent manner, as reported in Journal of the National Cancer Institute 78:149-154, 1987. Treatment of rats with ozone increases the production of nitric oxide by inducible nitric oxide synthetase of pulmonary macrophages, and more nitric oxide was produced in rats treated with interferon gamma, as reported in Journal of Immunology 151:7196-7205, 1993. Synthesis of nitric oxide from arginine is believed to be responsible for the tumoricidal effect of activated macrophages, as reported in Cancer Research 50:1421-1425, 1990.
One aspect of the present invention relates to enhancing biological activity of thioretinaco by reaction with ozone and oxygen to form a thioretinaco ozonide disulfonium complex.
Because of its lipophilic thioretinamide groups, thioretinaco is bound to the lipid bilayer of normal cells, contains tetraene conjugated double bond systems and a carbonyl group adjacent to the nitrogen atom of homocysteine thiolactone, and forms an octahedral complex with the cobalt atom of cobalamin, as reviewed in Annals of Clinical and Laboratory Science 24:27-59, 1994.
In accordance with the present invention, thioretinaco is believed to participate in oxidative phosphorylation in normal cells by formation of thioretinaco ozonide disulfonium complexes with ozone, oxygen and adenosine triphosphate within mitochondrial membranes. According to this concept, electrons from electron transport particles and protons from F.sub.1 F.sub.0 complexes, in the presence of dehydroascorbate, successively reduce the oxygen molecule that is bound to thioretinaco ozonide, catalyzing the stereospecific binding and release of adenosine triphosphate from the ATP synthetase of F.sub.1 complexes. The normal function of thioretinaco ozonide in oxygen metabolism is to limit the oxidative damage by reactive oxygen radicals to cellular macromolecules. The loss of thioretinaco ozonide from the membranes of neoplastic and senescent cells is believed to explain the increased oxidative damage that is found in the macromolecular constituents of these cells.
Another important function of thioretinaco ozonide in normal cells is believed to be the stereospecific synthesis of adenosyl methionine from methionine and adenosine triphosphate that is bound to the ATP synthetase of F.sub.1 complexes of mitochondrial membranes, as reviewed in Annals of Clinical and Laboratory Science 24:134-152, 1994. During cell division and growth of normal tissues, thioretinaco is believed to be reversibly converted to thioco, increasing intracellular free radical oxidants, and oxidizing glutathione and ascorbate. In this process, reactive oxygen species, such as superoxide, oxidize the sulfur atom of homocysteine thiolactone to sulfate, the precursor of the coenzyme, phosphoadenosine phosphosulfate, that sulfates the glycosaminoglycans of connective tissues during tissue growth.
The efficiency of homocysteine thiolactone metabolism declines with aging, explaining increased synthesis of homocysteine thiolactone, increased serum homocysteine concentration, and decreased serum adenosyl methionine during aging. These observations suggest and it is believed that thioretinaco ozonide is gradually lost from cellular membranes during the aging process. It is further believed, according to this concept, that the increasing risk of atherogenesis and carcinogenesis with aging is related to increased susceptibility to loss of thioretinaco ozonide by atherogenic factors and by carcinogenic factors.
Another aspect of the present invention also relates to further enhancement of biological activity of thioretinaco ozonide/thioretinaco ozonide disulfonium complex with the cytokines, alpha-, beta-, and gamma-interferons. The substantially enhanced anticarcinogenic, antineoplastic, antiviral, antiatherogenic, and antiaging activities of thioretinaco are believed to occur by specific stabilization and binding of thioretinaco ozonide by interferon within the membranes of treated cells. It is believed, according to this concept of my invention, that the concentration and activity of thioretinaco ozonide, combined with interferon, are increased within cell membranes. Because of this effect, the synthesis of adenosyl methionine from methionine is increased, and the synthesis of homocysteine thiolactone from methionine is decreased. Thus, treatment in accordance with the present invention, reverses the biochemical abnormalities in neoplasia, atherosclerosis, viral infections, autoimmune diseases, and aging, accounting for the enhanced biological activities of thioretinaco ozonide interferon complexes. In accordance with the present invention, the accumulation of reactive oxygen radicals is diminished, the thiolation of proteins, nucleic acids, and glycosaminoglycans by homocysteine thiolactone is diminished, thioco is converted to thioretinaco, diminishing the growth rate of affected cells, oxidative phosphorylation is increased, reversing the biochemical abnormalities of neoplastic cells, myointimal cells of atheromas, virally infected cells, and senescent cells. These multiple biochemical effects are believed to explain the enhanced anticarcinogenic, antineoplastic, antiviral and antiaging effects of thioretinaco ozonide by interferons, as taught in the present invention.